Development of a Fluorescent-Bead-Based Multiplex Immunoassay To Determine Immunoglobulin G Subclass Responses to Neisseria meningitidis Serogroup A and C Polysaccharides

Abstract

A fluorescent-particle-based multiplex flow cytometric immunoassay (MIA) for the detection of serum immunoglobulin G (IgG) and two IgG subclasses, IgG1 and IgG2, specific for Neisseria meningitidis serogroup A (MenA) and C (MenC) polysaccharides (PS) was developed. The assay comprised three separate duplex assays, one for the detection of the IgG response to MenA and MenC PS, another for the detection of the IgG1 response to MenA and MenC PS, and a third for the detection of the IgG2 response to MenA and MenC PS. Next, the three separate duplex assays were combined and analyzed as a hexaplex assay. No interference between monoplex, duplex, and hexaplex assays was observed, and the assay was found to have low intra-and interassay variation (<9.0% and <27%, respectively). Comparison of the meningococcal subclass MIA to the in-house enzyme-linked inmmunosorbent assays showed a good correlation (R > 0.85) for each of the subclasses. We conclude that the hexaplex meningococcal subclass MIA is an easy and specific assay for the determination of anti-MenA and anti-MenC PS subclass IgG, requiring minimal amounts of serum to study IgG subclass responses to vaccines.

Development of a Bead Immunoassay To Measure Vi Polysaccharide-Specific Serum IgG after Vaccination with the Salmonella enterica Serovar Typhi Vi Polysaccharide

“…Of 168 assays performed, a total of 13 (7.7%) were repeated due to the assays failing our QC guidelines. The interassay coefficient of variation for our control sample was 15.44% and is similar to what others have reported for bead immunoassays (9 to 27% [8]) and similar to what has been reported as acceptable performance for ELISA (37).…”

“…Our Vi polysaccharide bead assay is similar in design to other bead assays that utilize polysaccharide antigens covalently conjugated to fluorescent assay beads (5,8,23,27,34,35). In our study, due to a much lower background signal, the Vi bead assay provided a signal to noise ratio at each standard dilution that was superior to the signal to noise ratio obtained by ELISA.…”

“…The fluorescent bead assays were comparable to ELISA and sometimes were noted as having enhanced dynamic ranges or increased sensitivity (5,8,27,35). An additional benefit of fluorescent bead immunoassays is their ability to be multiplexed to permit the simultaneous measurement of antibodies specific for different antigens (8,23,27,34,35). This study was performed to evaluate a fluorescent bead immunoassay for its ability to measure vaccine-induced antibodies specific for Salmonella serotype Typhi Vi polysaccharide.…”

“…Immunoassays based on the use of fluorescent beads as the solid surface have recently been developed and compared to ELISA for the measurement of antigen-specific antibodies for polysaccharides from Streptococcus pneumoniae, Neisseria meningitidis, or Haemophilus influenzae type b (HiB) (5,8,23,27,34,35). The fluorescent bead assays were comparable to ELISA and sometimes were noted as having enhanced dynamic ranges or increased sensitivity (5,8,27,35).…”

Development of a Multi-Plex Electrochemiluminescent Assay for the Detection of Serum Antibody Responses to Meningococcal Conjugate Vaccines

“…Compared to traditional ELISA methods, ECL-based assays are highly sensitive, reproducible, robust, faster, and require lower sample volumes [8,9]. Recently, several groups have used a fluorescent bead based approach to develop multi-plex assays to analyze antibody responses to N. meningitidis [10,11]. However, limitations remain for this type of assay, including a requirement for covalent conjugation of polysaccharides to beads, which may impact polysaccharide conformation, and reliance on fluidic based measurement techniques which are intrinsically slow and prone to clogging.…”